The immune response is coordinated by the actions of cytokines produced from activated T lymphocytes, such as lymphocytes contacted with antigens. Cytokines are responsible for the control of proliferation and cell fate decisions among precursors of B cells, granulocytes and macrophages. T lymphocytes having a broad spectrum of antigen receptors are produced in the thymus as a product of the processes of selection and differentiation. When these T cells migrate to the peripheral lymphoid organs and encounter antigen, they undergo activation, during the process of which they produce large numbers of cytokines that act upon other cells of the immune system to coordinate their behavior to bring about an effective immune response.
T lymphocyte activation involves the specific regulation of many genes from minutes after the antigen encounter until at least 10 days later. T cells may also be activated by stimuli such as the combination of a calcium ionophore (e.g., ionomycin) and an activator of protein kinase C, such as phorbol myristate acetate (PMA). Several lectins, including phytohemagglutinin (PHA) may also be used to activate T cells (Nowell, P. C. (1990) Cancer Res. 20:462-466). The T cell activation genes are roughly grouped based on the time after stimulation at which each gene is regulated. Early genes trigger the regulation of subsequent genes in the activation pathway.
Because of the critical role of the T lymphocyte, agents that interfere with the early activation genes, such as cyclosporin A and FK506, are effective immunosuppressants. These early activation genes are regulated by transcription factors, such as NF-AT, that in turn are regulated through interactions with the antigen receptor. These transcription factors act through enhancer and promoter elements on the early activation genes to modulate their rate of transcription.
A typical early gene enhancer element is located in the first 325 base pairs upstream of the start site of the interleukin-2 gene. This region has been used extensively to dissect the requirements for T lymphocyte activation. This region binds an array of transcription factors including NF-AT, NFkB, Ap-1, Oct-1, and a newly identified protein that associates with Oct-1 called OAP-40. These different transcription factors act together to integrate the complex requirements for T lymphocyte activation.
The interleukin-2 gene is essential for both the proliferation and immunologic activation of T cells. The signaling pathways which connect the IL-2 gene and a representative and important early gene with the antigen receptor on the T cell surface and the signal transmission pathways between them are illustrated in FIG. 1. The binding site for the NF-AT protein appears to restrict expression of the interleukin-2 gene and other early activation genes to the context of an activated T lymphocyte. This information is based upon past work by Durand et al., Mol. and Cell. Biol. (1988), Shaw et al., Science, 241: 202 (1988), and Verwiej et al., (1990) J. Biol. Chem, 265: 15788-15795 (1990).
NF-AT appears to be the most important element among the group mentioned above in that it is able to direct transcription of any genes to activated T cells in the context of an intact transgenic animal (Verweij et al. J. Biol. Chem. 265:15788-15795, 1990). NF-AT is also the only element that requires physiologic activation through the antigen receptor for the activation of transcription by NF-AT. For example, the element is activated only after proper presentation of antigen of exactly the correct sequence by MHC-matched antigen presenting cells. This effect can be mimicked by pharmacologic agents, including the combination of ionomycin and PMA, which can also activate T cells through critical early genes.
Other elements within the IL-2 enhancer, for example, the NF-kB site or the AP-1 site, activate transcription in response to less specific stimuli, such as tumor necrosis factor alpha or simply PMA by itself. These compounds do not activate the IL-2 gene and other early activation genes and do not lead to T cell activation. Such observations have led to the conclusion that NF-AT restricts the expression of certain early genes, such as the interleukin-2 gene to their proper biologic context. Preliminary data have also indicated that a selective genetic deficiency of NF-AT produces severe combined immunodeficiency (SCID) (Chatilla, T. et al. New Engl. J. Med. 320:696-702, 1989).
As noted above, cyclosporin A (CsA) and FK506 are capable of acting as immunosuppressants. These agents inhibit T and B cell activation, mast cell degranulation and other processes essential to an effective immune response (Borel et al. (1976) Agents Actions 6: 468; Sung et al. (1988) J. Exp. Med. 168: 1539; Gao et al. Nature 336: 176). In T lymphocytes, these drugs disrupt an unknown step in the transmission of signals from the T cell antigen receptor to cytokine genes that coordinate the immune response (Crabtree et al. (1989) Science 243: 355; Schreiber et al. (1989) Science 251:283; Hohman & Hutlsch (1990) New Biol. 2: 663).
Putative intracellular receptors for FK506 and CsA have been described and found to be cis-trans prolyl isomerases (Fischer & Bang (1985) Biochim. Biophys. Acta 828: 39; Fischer et al. Nature 337: 476; Handschumacher et al. (1984) Science 226: 544; Lang & Schmid (1988) Nature 331: 453; Standaert et al. (1990) Nature 346: 671). Binding of the drugs inhibits isomerase activity; however, studies with other prolyl isomerase inhibitors (Bierer et al. (1990) Science 250: 556) and analysis of cyclosporin-resistant mutants in yeast suggest that the prevention of T lymphocyte activation results from formation of an inhibitory complex involving the drug and the isomerase (Bierer et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87: 9231; Tropschug et al. (1989) Nature 342: 953), and not from inhibition of the isomerase activity per se.
The transcription factor NF-AT appears to be a specific target of cyclosporin A and FK506, since transcription directed by this protein is completely blocked in T cells treated with these drugs, with little or no effect on other transcription factors, such as AP-1 and NF-KB (Shaw et al.(1988) op.cit; Emmel et al. (1989) Science 246: 1617; Mattila et al. (1990) EMBO J. 9: 4425). However, the drugs' actual mechanism of action remains unclear. Unfortunately, while both are potent immunosuppressive agents, neither cyclosporin nor FK506 are ideal drugs.
For example, cyclosporin adverse reactions include renal dysfunction, tremors, nausea and hypertension. Indeed, for many years researchers have attempted to develop superior replacements, with FK506 being the most recent candidate. Without understanding how cyclosporin (or FK506) functions at the intracellular level, developing improved immunosuppressants represents an extremely difficult research effort with a very limited likelihood of success.
Thus, there exists a significant need to understand the functional basis of cyclosporin and FK506 effectiveness. With such knowledge, improved assays for screening drug candidates would become feasible, which could in turn dramatically enhance the search process. The present invention fulfills these and other needs.